## Get DTT (in the biology section of the desiccator in the fridge--which is on the second shelf from bottom right side).

## Get DTT (in the biology section of the desiccator in the fridge--which is on the second shelf from bottom right side).

## Mix 54mg of DTT in 950µL of loading dye.

## Mix 54mg of DTT in 950µL of loading dye.

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# Determine amount of protein and buffer in each lane according to following table: Note that the protein amount ''CANNOT exceed 8µg per lane for 10 lane gel, and 6µg per lane for 15 lane gel.'' Get 1.5mL microtubes as many as needed.

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# Determine amount of protein and buffer in each lane according to following table: Note that the protein amount ''CANNOT exceed 8µg per lane for 10 lane gel, and 6µg per lane for 15 lane gel.'' Get 1.5mL microtubes as many as needed. [[Image:GelLoading.png | center | 400 px | Gel loading conditions]]

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[[Image:GelLoading.png | center | 400 px | Gel loading conditions]]

# Heat the tubes for 5-10 min, and get the ladder (marked "P" at the top in the Alaska fridge, in the box "Joshi Gel ladders/dyes") out to thaw.

# Heat the tubes for 5-10 min, and get the ladder (marked "P" at the top in the Alaska fridge, in the box "Joshi Gel ladders/dyes") out to thaw.

Mix gently by pipetting up and down or flicking the tube 4-5 times. Do not vortex. Place the mixture on ice for 30 minutes. Do not mix.

Heat shock at 42°C for 30 seconds. Do not mix.

Transfer tubes on ice for 2 minutes.

Add 950 µl of room temperature SOC media to tubes.

Place the tube at 37°C for 60 minutes. Shake vigorously (250 rpm) or rotate.

Warm selection plates to 37°C.

Spread 100 µl of the cells onto the plates with appropriate antibiotics. Use amp plates for positive control sample.

Incubate plates overnight at 37°C.

Qiagen MiniPrep

For overnight cell cultures 1~5mL of E. Coli in LB medium:

Transfer cell culture into centrifuge tubes

Centrifuge for 5 min. at 4,000 rpm

Discard the supernatant

Resuspend pelleted bacterial cells in 250 µl Buffer P1 and transfer to a microcentrifuge tube. Ensure that RNase A has been added to Buffer P1. No cell clumps should be visible after resuspension of the pellet.

Add 250 µl Buffer P2 and mix thoroughly by inverting the tube 4–6 times. Do not vortex. Continue inverting the tube until the solution becomes viscous + slightly clear blue. Do not allow lysis reaction to proceed for more than 5 min.

Add 350 µl Buffer N3 and mix immediately + thoroughly by 4-6x inverting the tube. The solution should become cloudy.

BIO-RAD Protein Gel

Get DTT (in the biology section of the desiccator in the fridge--which is on the second shelf from bottom right side).

Mix 54mg of DTT in 950µL of loading dye.

Determine amount of protein and buffer in each lane according to following table: Note that the protein amount CANNOT exceed 8µg per lane for 10 lane gel, and 6µg per lane for 15 lane gel. Get 1.5mL microtubes as many as needed.

Heat the tubes for 5-10 min, and get the ladder (marked "P" at the top in the Alaska fridge, in the box "Joshi Gel ladders/dyes") out to thaw.

While the tubes are being heated, prepare the gel:

Take the green cap off the wells and clean the wells with water. Repeat washing about three times. Take the tape off bottom.

Assemble the cassette with two gels or (one gel + a gel-wall). Note that the wells should face inwards.

Put the cassette into the gel box and pour NEW Tris-gly-SDS buffer into the cassette past. The buffer level should be past the wells. Check for any leaking. If there is a leak, reassemble the cassette and try again.

Set up the voltage machine to the desired voltage and time. For a fast run, 190V with 35 minutes could work. For slow and cleaner result, 90V with 50min could work.

Now get the heated samples, and the ladder. Add 10µL of ladder for 15 lane gels, 15µL of ladder for 10 lane gels. Add 15µL of the sample for 15 lane gels, 30µL for of the 10 lane gels.

Fill the box to the 2-gel mark (or 4-gel mark if running more than 3 gels) or more with OLD Tris-gly-SDS buffer. Put the cap on and hit the run button. Check for bubbles rising.

Coomassie Staining Protocol

\begin{enumerate}

\item Dump the Tris-gly-SDS buffer into the ``used" bottle.

\item Disassemble the cassette, set the gel down on a paper towel, and rinse the rest.

\item Fetch a box to put the gel in. Crack the gel open on all four corners and retrieve the gel; be careful not the rip, and if sticky apply water. Put it in the box.

\item Check the staining solution (50\% methanol, 40\% water, 10\% acetic acid, 0.25mg per 100mL) in the fume-hood in the back corridor.

\item If the caps have been open, or the solution is out, make a new one (500mL) by:

\begin{enumerate}

\item 50\% of total volume is methanol (is in the flammable cabinet in the chemistry room). 40\% of total volume is water. Use plastic graduated cylinder for these.

\item 10\% of total volume is acetic acid (is in the acid cabinet). BE CAUTIOUS WHEN WALKING CORNERS, and switches gloves after touching acid. Use GLASS graduated cylinder for this.

\item measure out and add the Coomassie to appropriate amount (0.25mg$\times$ total volume). It is located in the ``Bahamas"

\item NOTE: methanol waste must go to the special waste been under bench (bench with the pH meter). If the waste bin is full, call the number.

\end{enumerate}

\item Pour the Coomassie stain into the box.

BLACaM Assays

Set up plate reader

Plate should be read at 405 nm

Shake plate for 2-3 s before reading

Calculate concentrations and amounts needed to reach a total reaction volume of 150 μL